JPH09274039A - Diagnostic apparatus making use of immunological technique - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a diagnostic apparatus by which the existence of an object to be detected can be judged simply by a method wherein a sample which exists in a liquid is reacted with a labeled antibody or the like and a filter which is used to sort a reacted substance from an unreacted substance according to a physical size is used. SOLUTION: In a diagnostic kit, a sample which exists in a liquid is reacted with an antibody which is bonded immunologically to the sample or with an antibody which is bonded immunologically to the sample and which is labeled. After that, a filter 2 which sorts a reacted substance from an unreacted substance according to a physical size is used, and the existence of the sample is detected or the sample is measured quantitatively. Then, when one kind of a polyclonal antibody or two or more kinds of monoclonal antibodies exist, an object to be detected can be detected, and a complicated selection means to combine antibodies is not required. In addition, the diagnostic kit is important in order to detect a human villous gonad stimulant hormone which is utilized especially as a marker for the diagnosis of a pregnant woman.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to diagnosis of biofunctional substances such as proteins. This diagnostic device can be used as a means for detecting a substance secreted in body fluid, and is particularly useful in the medical field.

[0002]

2. Description of the Related Art Heretofore, as a method for detecting a non-detection substance by using an antibody, there are methods such as an enzyme immunoassay method, a latex agglutination method and an immunochromatography graph method using a Sandwich method. In particular, a method often used in recent years is immunochromatography using the sandwich method, which can be detected more easily than other methods. This method utilizes at least two types of antibodies, one of which is movable and labeled in a liquid phase. The other is permanently immobilized on the chromatographic carrier. When the sample is added, the labeled antibody first reacts with the sample, and then the bound antibody moves in the chromatographic carrier and is trapped there when it reaches the immobilization section. Many patent applications have been filed for this method, and, for example, Unilever application (Japanese Patent Laid-Open Nos. 5-50881, 5-150882, 2-503925), Abbott application (Japanese Patent Laid-Open No. 62-242872), Applied for high technology (JP-A-60-502364)
) Or a Berling Welke application (Japanese Patent Laid-Open No. 60-279418).

[0003]

In the conventional method, a sandwich method is used when detecting an object to be detected such as hCG. This method requires at least two or more types of monoclonal antibodies and their combination is important. Further, in the case of using a polyclonal antibody, one or more kinds of monoclonal antibodies are additionally required and their combination is also important. In order to select such a combination, enzyme-linked immunosorbent assay (ELISA) is generally used, but if a good combination cannot be found, there is a drawback that it is necessary to start producing an antibody again. It was

[0004]

The present invention relates to a sample (measurement target such as hCG) existing in a liquid, an antibody immunologically bound to the sample, or the sample. Equipped with a filter to separate the reaction product and unreacted product by physical size after reacting with immunologically bound and labeled antibody, and detect or quantitatively measure the presence or absence of sample The diagnostic kit is characterized in that one kind of polyclonal antibody or two
If there are more than one type of monoclonal antibody, the detection target can be detected. As a result, the sample as the detection target can be detected without taking a complicated selection means such as a combination of antibodies.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION By utilizing the diagnostic kit according to the present invention, it becomes possible to obtain a diagnostic kit for an object to be measured such as hCG without selecting a combination of the obtained antibodies.

Using two types of monoclonal antibody or one type of polyclonal antibody and a filter having a pore size of 10 nm or more, preferably a colored filter, hCG, CRP, LH, GH, IgG, CEA, αFP, LDH, FS
H, TSH, LH-RH can be detected.

Further, as shown in FIG. 2, a hCG, CRP, LH, GH, IgG, CEA, αFP, LDH, a diagnostic kit having a member for actively removing and absorbing a solution containing an unreacted substance was introduced. FSH, TSH,
LH-RH can be detected.

Further, by using a labeled antibody, particularly preferably a dye or enzyme-labeled antibody, it is possible to detect with high sensitivity regardless of the color of the filter. In particular, a diagnostic kit for detecting human chorionic gonadotropin, which is used as a marker for pregnancy diagnosis, is important, and will be described in detail below as an example.

[Preparation of antibody] (Immune) Purified hCG (purity: 98% or more, SDS electrophoresis assay)
Adjusted to 2 mg / ml using BS, and an equal volume of this (complete Freund's agent containing human tuberculosis killed bacteria, Wako Pure Chemical Industries, H37Rv)
Was added and well emulsified with a homogenizer (1000 rpm). Twenty mice (A / J) about 8 weeks old were injected with 100 μL each of the adjuvant emulsion containing the immunogen. Three weeks later, incomplete Freund's adjuvant having the same volume as the 2 mg / ml hCG solution was emulsified with a homogenizer, and 100 μl of this emulsion was injected into A / J mice.

Then, after 6, 9 and 12 weeks, 100 μl each of the incomplete Freund's adjuvant emulsion containing hCG was again injected into the mouse.
Blood was collected one week after the injection and the antibody production shown below was confirmed.

(Confirmation of antibody production) The collected serum was confirmed for antibody production by the enzyme-linked immunosorbent assay (ELISA) shown below. As the solid phase, 100 μl / well of hCG adjusted to 2.5 μg / ml with 0.1 mg / ml BSA / PBS / Az was used.

As a secondary antibody, peroxydase-labeled anti-mouse IgG
An antibody or a peroxidase-labeled anti-mouse IgM antibody was used.

As a result, the production of anti-hCG antibody was confirmed in all mice, and it was confirmed that the IgG / IgM ratio was 100 or more and class switching occurred.

(Cell fusion) Of the immunized mice, 2 mice with particularly high titers were boosted (injection of weak immunogen) in order to enlarge the spleen of the mice. As the immunogen, a 1 mg / mL hCG solution was used as it was without adding an adjuvant.

Spleen cells of the mouse 3 days after the boosting were extracted and fused with a mouse myeloma-derived cell line (P3X63-Ag8.653) by a conventional method using polyethylene glycol having an average molecular weight of 1,500. Spleen cells of the same mouse were used as phyta (cells that supply growth factors), and after culturing for 1 day in an Ishkov medium containing 15% fetal calf serum (FCS) on two 96-well plates (100
100 μl / well of HAT medium (100 μl / well) was added, and the cells were cultured in a CO 2 incubator (CO 2 concentration: 5%, temperature: 37 ° C., humidity: 95%) for 1 week. Then, after removing the culture supernatant, contains 15% FCS
The medium was replaced with HT medium (250 μl / well).

(Cell selection) One week after replacement with HT medium, 200 μl / well of the culture supernatant was taken out. HT medium containing 15% FCS (200 μl / well) was added and cultured for 3 days.
μl / well was taken out. A total of 400 μl / well of culture supernatant could be obtained.

Using this culture supernatant, the binding ability to hCG, hCG-α and hCG-β was measured by the following ELISA method.

0.1 mg / ml BSA / PBS / A to 2.5 μg / ml as a solid phase
100 μl / well of hCG, hCG-α, and hCG-β adjusted with z were used. The cell culture supernatant was used as the antibody solution.

As a result, 2 wells showed binding ability only to hCG and hCG-α, and 2 wells showed binding ability only to hCG and hCG-β. In addition, 4 wells showed binding ability to all hormones.

(Cloning) The cells in the above 8 wells were diluted (limit dilution) to a concentration containing 1 cell per well, and dispensed into 8 96-well microplates. The initial proliferation was promoted by using the thymocytes of 5-week-old mice (Balb / c) as a feeder. Continue the culture while increasing the plate size, and EL
By repeating screening by the ISA method, a cell line showing a high titer against hCG and showing good growth was finally selected, and the culture was advanced to a concentration of 5 × 10 5 cells / mL in 200 ml. .

As a result, one strain showing a binding ability only to hCG and hCG-α and having a high affinity for hCG, and hC
One strain was selected that showed binding ability only to G and hCG-β and had a high affinity for hCG.

(Preservation of cells) For the finally selected cell line, the supernatant was centrifuged and suspended in 1 mL of a solution of FCS: dimethyl sulfoxide = 9: 1 at a concentration of 5 × 10 6 cells / mL. After freezing at -80 ℃, it was transferred to -135 ℃ for long-term storage.

(Purification of antibody) The selected two strains were subjected to large-scale culture, and the supernatant was isolated by centrifugation. Regarding the cell culture supernatant of each strain, protein A binding gel (protein A Sepharose CL
Each monoclonal antibody was purified from the cell culture supernatant by affinity chromatography using -4B (Pharmacia). This monoclonal antibody was confirmed by SDS polyacrylamide gel electrophoresis to be a purified antibody as an IgG composed of an H chain of about 50,000 and an L chain of about 25,000 by comparison with a standard protein.

(Evaluation of Antibodies) The two types of monoclonal antibodies purified by the above affinity chromatography were subjected to antibody evaluation by the following ELISA method.

As a result, the binding ability to hCG was measured for one strain (cell line name: MN-α1) that exhibited binding ability only to hCG and hCG-α. The binding ability to hCG was measured for one strain (cell line name: MN-β3) that exhibited binding ability only to hCG and hCG-β.

MN-α1 was able to detect hCG at a concentration higher than 10-9M. Furthermore, MN-β3 was also able to detect hCG at a concentration higher than 10-9M.

Although the data are not shown here, when the binding ability to each hormone was measured again for each cell line,
MN-α1 showed binding ability to hCG and hCG-α. On the other hand, MN-β3 showed binding ability to hCG and hCG-β, but did not show binding ability to hCG-α.

(Enzyme-linked immunosorbent assay (ELISA)) The obtained antisera, culture supernatants and monoclonal antibodies were evaluated. The operation method is described below.

(A) Antigen coating hCG, hCG-α and hCG-β in 0.1 mg / ml BS so that the coating becomes 2.5 μg / ml
Adjusted with A / PBS / Az. 100 μL / well of the antigen solution was injected into a microplate (96-well plate made by vinyl chloride Costar) and stored at 20 ° C. overnight. The antigen solution was removed with an aspirate.

(B) Blocking BSA / PBS / Az was injected at 250 μL / well and left at room temperature for 30 minutes. After that, BSA / PBS / Az was removed with an aspirate. When no subsequent experiments were performed on the same day, they were stored in this state at 4 ° C. together with filter paper moistened with water.

(C) Antibody reaction 50 μL / well of antibody solution (antiserum, culture supernatant, purified antibody, etc.) diluted with 1% BSA / PBS / Az to various dilution ratios and 1% BSA / PBS / A
Z50 μL / well was injected. Inhibition solution (hCG, hCG-
α, hCG-β) 50 μL / well was injected, and 50 μL / well of the antibody solution was further added while shaking. After storing at room temperature for 3 hours, the antibody solution was removed with an aspirate and washed 3 times with PBS to remove the remaining PBS with an aspirate.

(D) Reaction of secondary antibody 0.2 μg / mL peroxydase-labeled anti-mouse IgG antibody derived from YAG (KPL
(Made by the company) dissolved in 1% BSA in PBS or 0.2μ
50 μL / well of g / mL peroxydase-labeled anti-mouse IgM antibody yak-derived (manufactured by KPL) dissolved in 1% BSA in PBS was injected and left at room temperature for 30 minutes. It was removed with an aseptor, washed three times with PBS, and the remaining PBS was removed with an aspirate.

(E) Substrate reaction and termination 40 mg of o-phenylenediamine (for biochemistry) was dissolved in 10 mL of citric acid-phosphate buffer (pH 5), and 4 μL of 30% hydrogen peroxide solution was added immediately before use. The solution (substrate solution) was injected at 100 μL / well and left at room temperature. Ten minutes later, the reaction was stopped by injecting 25 μL / well of 4N sulfuric acid.

(F) Measurement The absorbance at 492 nm or above was measured using a Toyo Sorter Microplate Reader. In the first column, pure water was usually injected to serve as a reference value, and a blank value where only the item (C) was omitted was used.

[Dnsyl labeling of hCG antibody] 500 mg (0.0076 mmol) of each of the obtained two kinds of anti-hCG monoclonal antibodies was dissolved in 20 ml of PBS and stirred at room temperature for 390 m.
1 ml of a PBS solution of g (0.38 mmol) of Dansyl chloride was added dropwise. After stirring at room temperature for 10 minutes, the mixture was left overnight at 4 ° C. The reaction solution is subjected to gel filtration (G-25M column, developing solution: P
BS) to obtain 40 ml of antibody solution. A glass fiber filter paper was impregnated with this labeled antibody solution and freeze-dried.

[Treatment of nitrocellulose filter] Nitrocellulose filter (S &
Spores (Phoasize 1 μm) is infiltrated with a 1% BSA / PBS solution for 30 minutes. After that, remove water well and wash while shaking with 100 ml of distilled water. Repeat this operation two more times. Then, it is dried at room temperature and left for 1 hour at a humidity of 35% or less. This nitrocellulose filter can be used for at least 3 months by storing it in a light-shielded state at a humidity of 35% or less.

[Production of Diagnostic Kit] The diagnostic kit has the configuration shown in FIG. At the bottom of the labeled antibody-impregnated filter paper 1 above,
Place 1 μm nitrocellulose filter 2. Further, a water absorbing member 3 for absorbing and removing unreacted substances is installed below the water absorbing member 3. These one
Members 3 to 3 are mounted in the container 4. Further, the labeled antibody-impregnated filter paper 1 can be moved left and right.

[Measurement of Human Chorionic Gonadotropin] From the upper part of the diagnostic kit shown in FIG.
μl (concentration 40000 IU / L) was added. After about 3 minutes, the labeled antibody-impregnated filter paper was slid to expose the nitrocellulose filter. As a result, the surface of the nitrocellulose filter was yellow. on the other hand,
After adding 500 μl of an aqueous solution containing no hCG, the impregnated filter paper was similarly slid. As a result, the nitrocellulose filter surface remained white.

[0039]

INDUSTRIAL APPLICABILITY As described above, the present invention provides a method for efficiently introducing the obtained antibody into a diagnostic kit.

Further, it becomes possible to easily determine the presence or absence of the detection object.

[Brief description of drawings]

FIG. 1 is a diagram showing a configuration of a diagnostic kit according to an embodiment of the present invention.

FIG. 2 is a diagram showing a configuration of a diagnostic kit according to an embodiment of the present invention.

FIG. 3 is a diagram showing the structure of a human chorionic gonadotropin diagnostic kit in one example of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location G01N 33/545 G01N 33/545 B 33/68 33/68 // G01N 33/577 33/577 A

Claims (9)

[Claims] 1. A sample present in a liquid is reacted with an antibody immunologically bound to the sample or an antibody immunologically bound to the sample and labeled, A diagnostic device comprising a filter for separating a reaction product and an unreacted product according to physical size, and detecting or quantitatively measuring the presence or absence of the sample. 2. A sample present in a liquid is reacted with an antibody immunologically bound to the sample or an antibody immunologically bound to the sample and labeled, A diagnostic device for detecting or quantitatively measuring the presence or absence of a sample by the physical size of a reaction product and an unreacted product, and a member containing the reaction product and capable of detecting the presence or absence of the sample and a solution containing the unreacted product A diagnostic device comprising a member for absorbing the. 3. A sample present in a liquid is reacted with an antibody immunologically bound to the sample or an antibody immunologically bound to the sample and labeled, A diagnostic kit for detecting or quantitatively detecting the presence or absence of a sample by the physical size of a reaction product and an unreacted product,
A diagnostic device comprising a member containing an antibody or a labeled antibody in a dried state, a member capable of detecting the presence or absence of a sample by holding a reaction product, and a member for absorbing a solution containing an unreacted product. . 4. The diagnostic device according to claim 1, 2 or 3, wherein the sample is a protein, a peptide, a nucleic acid, a polysaccharide or a lipid which is a biofunctional substance. 5. The diagnostic apparatus according to claim 1, 2 or 3, wherein the antibody is a monoclonal antibody, a polyclonal antibody, a chimeric antibody, or a product obtained by enzymatically treating these antibodies. 6. The particle is a granular polymer such as an enzyme, a fluorescent substance, a dye, a dye gel, a metal colloid, a metal oxide colloid, or a latex, and the label is characterized in that:
A diagnostic device as described. 7. The diagnostic apparatus according to claim 1, 2 or 3, wherein the filter has a pore size of 10 nm or more and 10 μm or less. 8. The sample protein is hCG, CRP, LH, GH, Ig
2. The diagnostic device according to claim 1, which is G, CEA, αFP, LDH, FSH, TSH, or LH-RH. 9. A chorionic gonadotropin present in a body fluid and an antibody that immunologically binds to the chorionic gonadotropin, or an antibody that immunologically binds to the chorionic gonadotropin. It is also a diagnostic kit that detects the presence or absence of a sample or measures quantitatively the physical size of the reaction product and unreacted product after reacting with a labeled antibody, and contains the antibody or the labeled antibody in a dry state. A pregnancy diagnosis apparatus comprising: a member capable of visually detecting the presence or absence of chorionic gonadotropin by holding a reaction member and a reaction member, and a member for absorbing a solution containing an unreacted material.